1. Field of the Invention
The present invention is directed the field of bacteriophages. Specifically, it is directed to a method and device for sanitation using a bacteriophage.
2. Description of Related Art
Vancomycin-resistant Enterococcus
Over the last ten years there has been an emergence of bacterial pathogens, which demonstrate resistance to many, if not all antimicrobial agents. This is particularly relevant in the institutional environment where nosocomial pathogens are under selective pressure due to extensive antimicrobial usage. A particular problem in this regard has been vancomycin-resistant enterococci (VRE), which are not treatable with standard classes of antibiotics. Despite the recent release of two drugs to which VRE are susceptible (quinupristin/dalfopristin and linezolid [Plouffe JF, Emerging therapies for serious gram-positive bacterial infections: A focus on linezolid. Clin Infect dis 2000 Suppl 4:S144-9), these microorganisms remain an important cause of morbidity and mortality in immunocompromised patients.
Enterococci are gram positive facultatively anaerobic cocci found in a variety of environmental sources including soil, food and water. They are also a common colonizing bacterial species in the human intestinal tract (i.e., the intestinal tract serves as a reservoir for the microorganism). Although the taxonomy of enterococci has not been finalized, it is generally accepted that the genus consists of 19 species.
Antibiotic management of serious enterococcal infections has always been difficult due to the intrinsic resistance of the organisms to most antimicrobial agents [Arden, R. C, and B. E. Murray, 1994, xe2x80x9cEnterococcus: Antimicrobial resistance.xe2x80x9d In: Principles and Practice of Infectious Diseases Update, volume 2, number 4 (February, 1994). New York: Churchill Livingstone, Inc. 15 pps; Landman, D., and J. M. Quale, 1997, xe2x80x9cManagement of infections due to resistant enterococci: a review of therapeutic options.xe2x80x9d J. Antimicrob. Chemother., 40:161-70; Moellering, R. C., 1998, xe2x80x9cVancomcyin-resistant enterococci.xe2x80x9d Clin. Infect. Dis. 26:1196-9]. In the 1970""s enterococcal infections were treated with the synergistic combination of a cell wall active agent such as penicillin and are aminoglycoside (Moellering, et al. (1971), xe2x80x9cSynergy of penicillin and gentamicin against enterococci.xe2x80x9d J Infect. Dis., 124:S207-9; Standiford, et al. (1970), xe2x80x9cAntibiotic synergism of enterococci: relation to inhibitory concentrations.xe2x80x9d Arch. Intern: Med., 126: 255-9). However, during the 1980""s enterococcal strains with high levels of aminoglycoside resistance and resistance to penicillin, mediated both by a plasmid-encoded xcex2-lactamase and by changes in penicillin binding proteins, appeared (Mederski-Samoraj, et al. (1983), xe2x80x9cHigh level resistance to gentamicin in clinical isolates of enterococci.xe2x80x9d J. Infect. Dis., 147:751-7; Uttley, et al. (1988), xe2x80x9cVancomycin resistant enterococci.xe2x80x9d Lancet i:57-8). In 1988 the first VRE isolates were identified (Leclercq, et al. (1988), xe2x80x9cPlasmid mediated resistance to vancomycin and teicoplanin in Enterococcus faecium.xe2x80x9d N Engl. J: Med., 319:157-61). Such organisms, called VRE because of resistance to vancomycin, are also resistant to the penicillin-aminoglyroside combination. VRE includes strains of several different enterococcal species with clinically significant VRE infections caused by Enterococcus faecium and Enterococcus faecalis. 
Enterococci can cause a variety of infections including wound infection, endocarditis, urinary tract infection and bacteremia. After Staphylococcus aureus and coagulase negative staphylococci, enterococci are the most common cause of nosocomial bacteremia. Among immunocompromised patients, intestinal colonization with VRE frequently precedes, and serves as a risk factor for, subsequent VRE bacteremia(Edmond, et al. (1995), xe2x80x9cVancomycin resistant Enterococcus faecium bacteremia: Risk factors for infection.xe2x80x9d Clin. Inf. Dis., 20:1126-33; Tornieporth, N. G., R. B. Roberts, J. John, A. Hafner, and L. W. Riley, 1996, xe2x80x9cRisk factors associated with vancomycin-resistant Enterococcus faecium infection or colonization in 145 matched case patients and control patients.xe2x80x9d Clin. Infect. Dis., 23:767-72.]. By using pulse field gel electrophoresis as a molecular typing tool investigators at the University of Maryland at Baltimore and the Baltimore VA Medical Center have shown VRE strains causing bacteremia in cancer patients are almost always identical to those which colonize the patients gastrointestinal tract (Roghmann M C, Qaiyumi S, Johnson J A, Schwalbe R, Morris J G (1997), xe2x80x9cRecurrent vancomycin-resistant Enterococcus faecium bacteremia in a leukemia patient who was persistently colonized with vancomycin-resistant enterococci for two years.xe2x80x9d Clin Infect Dis 24:514-5). The risk of acquiring VRE increases significantly when there is a high rate of VRE colonization among patients on a hospital ward or unit (i.e., when there is high xe2x80x9ccolonization pressurexe2x80x9d). In one study in the Netherlands, colonization pressure was the most important variable affecting acquisition of VRE among patients in an intensive care unit (Bonten M J, et al, xe2x80x9cThe role of xe2x80x9ccolonization pressurexe2x80x9d in the spread of vancomycin-resistant enterococci: an important infection control variable.xe2x80x9d Arch Intern Med 1998;25:1127-32). Use of antibiotics has been clearly shown to increase the density, or level of colonization, in an individual patient (Donskey C J et al, xe2x80x9cEffects of antibiotic therapy on the density of vancomycin-resistant enterococci in the stool of colonized patients.xe2x80x9d N Engl J Med 2000;343:1925-32): this, in turn, would appear to increase the risk of subsequent infection, and the risk of transmission of the organism to other patients.
Multi-Drug Resistant Staphylococcus aureus (MDRSA)
S. aureus is responsible for a variety of diseases ranging from minor skin infections to life-threatening systemic infections, including endocarditis and sepsis [Lowy, F. D., 1998, xe2x80x9cStaphylococcus aureus infections.xe2x80x9d N. Engl. J. Med, 8:520-532]. It is a common cause of community- and nosocomially-acquired septicemia (e.g., of approximately 2 million infections nosocomially acquired annually in the United States, approximately 260,000 are associated with S. aureus [Emori, T. G., and R. P. Gaynes, 1993, xe2x80x9cAn overview of nosocomial infections, including the role of the microbiology laboratory,xe2x80x9d Clin. Microbiol. Rev., 4:428-442]). Also, approximately 20% of the human population is stably colonized with S. aureus, and up to 50% of the population is transiently colonized, with diabetics, intravenous drug users, patients on dialysis, and patients with AIDS having the highest rates of S. aureus colonization [Tenover, F. C., and R. P. Gaynes, 2000, xe2x80x9cThe epidemiology of Staphylococcus infections,xe2x80x9d p. 414-421, In: V. A. Fischetti, R. P. Novick, J. J. Ferretti, D. A. Portnoy, and J. I. Rood (ed), Gram-positive pathogens, American Society for Microbiology, Washington, D.C.]. The organism is responsible for approximately one-half of all skin and connective tissue infections, including folliculitis, cellulitis, furuncules, and pyomyositis, and is one of the most common causes of surgical site infections. The mortality rate for S. aureus septicemia ranges from 11 to 48% [Mortara, L. A., and A. S. Bayer, 1993, xe2x80x9cStaphylococcus aureus bacteremia and endocarditis. New diagnostic and therapeutic concepts.xe2x80x9d Infect. Dis. Clin. North. Am., 1:53-68].
Methicillin was one of the first synthetic antibiotics developed to treat penicillin-resistant staphylococcal infections. However, the prevalence of methicillin-resistant S. aureus strains or xe2x80x9cMRSAxe2x80x9d (which also are resistant to oxacillin and nafcillin) has drastically increased in the United States and abroad [Panlilio, A. L., D. H. Culver, R. P. Gaynes, S. Banerjee, T. S. Henderson, J. S. Tolson, and W. J. Martone, 1992, xe2x80x9cMethicillin-resistant Staphylococcus aureus in U.S. hospitals, 1975-1991.xe2x80x9d Infect. Control Hosp. Epidemiol., 10:582-586]. For example, according to the National Nosocomial Infections Surveillance System [National Nosocomial Infections Surveillance (NNIS) report, data summary from October 1986-April 1996, issued May 1996, xe2x80x9cA report from the National Nosocomial Infections Surveillance (NNIS) System.xe2x80x9d Am. J. Infect. Control., 5:380-388], approximately 29% of 50,574 S. aureus nosocomial infections from 1987 to 1997 were resistant to the xcex2-lactam antibiotics (e.g., oxacillin, nafcillin, methicillin), and the percent of MRSA strains among U.S. hospitals reached approximately 40% by the end of the same period. At the University of Maryland Medical Center,  greater than 50% of all S. aureus blood isolates are now methicillin resistant.
In this setting, there is great concern about the possible emerge of methicillin-resistant/multi-drug resistant S. aureus strains which are vancomycin resistantxe2x80x94and which would be essentially untreatable. Although overt resistance to vancomycin has not yet been documented in clinical isolates, there have been several reports of clinical infections with S. aureus strains having intermediate resistance to vancomycin (MICs=8 xcexcg/ml), which suggests that untreatable staphylococcal infections may not be too far away [Tenover, F. C., and R. P. Gaynes. 2000]. Given the virulence of S. aureus, the emergence of such untreatable strains would be devastating and have a major impact on the way in which medicine is practiced in this country.
Staphylococcal species, including MDRSA, are common colonizers of the human nose; in one community-based study, 35% of children and 28% of their guardians had nasal Staphylococcus aureus colonization (Shopsin B, et al, xe2x80x9cPrevalence of methicillin-resistant and methicillin-susceptible Staphylococcus aureus in the community.xe2x80x9d J Infect Dis 2000;182:359-62.). Persons who are nasally colonized with MRSA have an increased risk of developing serious systemic infections with this microorganism, and, in particular, colonization or prior infection with MDRSA significantly increases the risk of subsequent bacteremia with MDRSA (Roghmann M C, xe2x80x9cPredicting methicillin resistance and the effect of inadequate empiric therapy on survival in patients with Staphylococcus aureus bacteremia. Arch Intern Med 2000;160:1001-4). As seen with VRE, the rate of colonization of persons with MDRSA on a unit (the colonization pressure) significantly increases the risk of acquisition of MDRSA for other patients on the unit (Merrer J, et al, xe2x80x9cxe2x80x9dColonization pressurexe2x80x9d and risk of acquisition of methicillin-resistant Staphylococcus aureus in a medical intensive care unit.xe2x80x9d Infect Control Hosp Epidemiol 2000;21:718-23).
Multi-drug Resistant Pseudomonas aeruginosa 
Pseudomonas aeruginosa is a highly virulent gram-negative bacterial species that is responsible for bacteremia, wound infections, pneumonia, and urinary tract infections. Increasing problems with multi-antibiotic resistance in Pseudomonas has been noted in hospitals, with particular concern focusing on strains which are generally designated as xe2x80x9cImipenem-resistant Pseudomonasxe2x80x9d, reflecting the last major antimicrobial agent to which they have become resistant. Many of these strains are resistant to all major antibiotic classes, presenting substantive difficulties in management of infected patients.
As seen with other Gram-negative microorganisms, Pseudomonas strains often emerge as the primary colonizing flora of the posterior pharynx during hospitalization. Strains present in the posterior pharynx, in turn, are more likely to be aspirated into the lungs, and cause pneumonia. In this setting, colonization with multi-drug resistant Pseudomonas represents a potentially serious risk factor for development of multi-drug resistant Pseudomonas pneumonia.
Bacteriophage
Bacteriophage has been used therapeutically for much of this century. Bacteriophage, which derive their name from the Greek word xe2x80x9cphagoxe2x80x9d meaning xe2x80x9cto eatxe2x80x9d or xe2x80x9cbacteria eatersxe2x80x9d, were independently discovered by Twort and independently by D""Herelle in the first part of the twentieth century. Early enthusiasm led to their use as both prophylaxis and therapy for diseases caused by bacteria. However the results from early studies to evaluate bacteriophage as antimicrobial agents were variable due to the uncontrolled study design and the inability to standardize reagents. Later in well designed and controlled studies it was concluded that bacteriophage were not useful as antimicrobial agents (Pyle, N. J. (1936), J. Bacteriol., 12:245-61; Colvin, M. G. (1932), J. Infect Dis., 51:17-29; Boyd et al. (1944), Trans R. Soc. Trop. Med. Hyg., 37:243-62).
This initial failure of phage as antibacterial agents may have been due to the failure to select for phage that demonstrated high in vitro lytic activity prior to in vivo use. For example, the phage employed may have had little or no activity against the target pathogen, were used against bacteria that were resistant due to lysogenization or the phage itself might be lysogenic for the target bacterium (Barrow, et al. (1997), xe2x80x9cBacteriophage therapy and prophylaxis: rediscovery and renewed assessment of potential.xe2x80x9d Trends in Microbiology, 5:268-71). However, with a better understanding of the phage-bacterium interaction and of bacterial virulence factors, it was possible to conduct studies which demonstrated the in vivo anti-bacterial activity of the bacteriophage (Asheshov, et al. (1937), Lancet, 1:319-20; Ward, W. E. (1943), J. Infect. Dis., 72:172-6; Lowbury, et al. (1953), J: Gen. Microbiol., 9:524-35). In the U.S. during the 1940""s Eli Lilly commercially manufactured six phage products for human use including preparations targeted towards staphylococci, streptococci and other respiratory pathogens.
With the advent of antibiotics, the therapeutic use of phage gradually fell out of favor in the U.S. and Western Europe and little subsequent research was conducted. However, in the 1970""s and 1980""s there were reports of bacteriophage therapy continuing to be utilized in Eastern Europe, most notably in Poland and the former Soviet Union.
Phage therapy has been used in the former Soviet Union and Eastern Europe for over half a century, with research and production centered at the Eliava Institute of Bacteriophage in Tbilisi, in what is now the Republic of Georgia. The international literature contains several hundred reports on phage therapy, with the majority of the publications coming from researchers in the former Soviet Union and eastern European countries. To give but a few examples, phages have been reported to be effective in treating (i) skin and blood infections caused by Pseudomonas, Staphylococcus, Klebsiella, Proteus, and E. coli [Cislo, M., M. Dabrowski, B. Weber-Dabrowska, and A. Woyton, 1987, xe2x80x9cBacteriophage treatment of suppurative skin infections,xe2x80x9d 35(2):175-183; Slopek, S., I. Durlakowa, B. Weber-Dabrowska, A. Kucharewicz-Krukowska, M. Dabrowski, and R. Bisikiewicz, 1983, xe2x80x9cResults of bacrteriophage treatment of suppurative bacterial infections. I. General evaluation of the results,xe2x80x9d Archivum. Immunol. Therapiae Experimental, 31:267-291; Slopek, S., B. Weber-Dabrowska, M. Dabrowski, and A. Kucharewicz-Krukowska, 1987, xe2x80x9cResults of bacteriophage treatment of suppurative bacterial infections in the years 1981-1986,xe2x80x9d, 35:569-83], (ii) staphylococcal lung and pleural infections [Meladze, G. D., M. G. Mebuke, N. S. Chkhetia, N. I. Kiknadze, G. G. Koguashvili, I. I. Timoshuk, N. G. Larionova, and G. K. Vasadze, 1982, xe2x80x9cThe efficacy of Staphylococcal bacteriophage in treatment of purulent diseases of lungs and pleura,xe2x80x9d Grudnaya Khirurgia, 1:53-56 (in Russian, summary in English)], (iii) P. aeruginosa infections in cystic fibrosis patients [Shabalova, I. A., N. I. Karpanov, V. N. Krylov, T. O. Sharibjanova, and V. Z. Akhverdijan. xe2x80x9cPseudomonas aeruginosa bacteriophage in treatment of P. aeruginosa infection in cystic fibrosis patients,xe2x80x9d abstr. 443. In Proceedings of IX international cystic fibrosis congress, Dublin, Ireland], (iv) neonatal sepsis [Pavlenishvili, I., and T. Tsertsvadze. 1985. xe2x80x9cBacteriophage therapy and enterosorbtion in treatment of sepsis of newbornes caused by gram-negative bacteria.xe2x80x9d In abstracts, p. 104, Prenatal and Neonathal Infections, Toronto, Canada], and (v) surgical wound infections [Peremitina, L. D., E. A. Berillo, and A. G. Khvoles, 1981, xe2x80x9cExperience in the therapeutic use of bacteriophage preparations in supportive surgical infections.xe2x80x9d Zh. Mikrobiol. Epidemiol. Immunobiol. 9:109-110 (in Russian)]. Several reviews of the therapeutic use of phages were published during the 1930s-40s [Eaton, M. D., and S. Bayne-Jones, 1934, xe2x80x9cBacteriophage therapy: review of the principles and results of the use of bacteriophage in the treatment of infections,xe2x80x9d J. Am. Med. Assoc., p. 103; Krueger, A. P., and E. J. Scribner, 1941, xe2x80x9cThe bacteriophage: its nature and its therapeutic use,xe2x80x9d J. Am. Med. Assoc., p. 116] and recently [Barrow, P. A., and J. S. Soothill, 1997, xe2x80x9cBacteriophage therapy and propylaxisxe2x80x94rediscovery and renewed assessment of potential,xe2x80x9d Trends in Microbiol., 5(7):268-271; Lederberg, J., 1996, xe2x80x9cSmaller fleas . . . ad infinitum: therapeutic bacteriophage,xe2x80x9d Proc. Natl. Acad. Sci. USA, 93:3167-3168]. In a recent paper published in the Journal of Infection (Alisky, J., K. Iczkowski, A. Rapoport, and N. Troitsky, 1998, xe2x80x9cBacteriophages show promise as antimicrobial agents,xe2x80x9d J. Infect., 36:5-15), the authors reviewed Medline citations (published during 1966-1996) of the therapeutic use of phages in humans. There were twenty-seven papers from Britain, the U.S.A., Poland and the Soviet Union, and they found that the overall reported success rate for phage therapy was in the range of 80-95%.
These are several British studies describing controlled trials of bacteriophage raised against specific pathogens in experimentally infected animal models such as mice and guinea pigs (See, e.g., Smith. H. W., and M. B. Huggins xe2x80x9cSuccessful treatment of experimental Escherichia coli infections in mice using phages: its general superiority over antibioticsxe2x80x9d J. Gen. Microbial., 128:307-318 (1982); Smith, H. W., and M. B. Huggins xe2x80x9cEffectiveness of phages in treating experimental E. coli diarrhea in calves, piglets and lambsxe2x80x9d J. Gen. Microbiol., 129:2659-2675 (1983); Smith, H. W. and R. B. Huggins xe2x80x9cThe control of experimental E. coli diarrhea in calves by means of bacteriophagexe2x80x9d. J. Gen. Microbial., 133:1111-1126 (1987); Smith, H. W., R. B. Huggins and K. M. Shaw xe2x80x9cFactors influencing the survival and multiplication of bacteriophages in calves and in their environmentxe2x80x9d J. Gen. Microbial., 133:1127-1135 (1987)). These trials measured objective criteria such as survival rates. Efficacy against Staphylococcus, Pseudomonas and Acinetobacter infections were observed. These studies are described in more detail below.
One U.S. study concentrated on improving bioavailability of phage in live animals (Merril, C. R., B. Biswas, R. Carlton, N. C. Jensen, G. J. Greed, S. Zullo, S. Adhya xe2x80x9cLong-circulating bacteriophage as antibacterial agentsxe2x80x9d Proc. Natl. Acad Sci. USA, 93:3188-3192 (1996)). Reports from the U.S. relating to bacteriophage administration for diagnostic purposes have indicated phage have been safely administered to humans in order to monitor humoral immune response in adenosine deaminase deficient patients (Ochs, et al. (1992), xe2x80x9cAntibody responses to bacteriophage phi X174 in patients with adenosine deaminase deficiency.xe2x80x9d Blood, 80:1163-71) and for analyzing the importance of cell associated molecules in modulating the immune response in humans (Ochs, et al. (1993), xe2x80x9cRegulation of antibody responses: the role of complement acrd adhesion molecules.xe2x80x9d Clin. Immunol. Immunopathol., 67:S33-40).
Additionally, Polish, Georgian, and Russian papers describe experiments where phage was administered systemically, topically or orally to treat a wide variety of antimicrobial resistant pathogens (See, e.g., Shabalova, I. A., N. I. Karpanov, V. N. Krylov, T. O. Sharibjanova, and V. Z. Akhverdijan. xe2x80x9cPseudomonas aeruginosa bacteriophage in treatment of P. aeruginosa infection in cystic fibrosis patients,xe2x80x9d Abstr. 443. In Proceedings of IX International Cystic Fibrosis Congress, Dublin, Ireland; Slopek, S., I. Durlakowa, B. Weber-Dabrowska, A. Kucharewicz-Krukowska, M. Dabrowski, and R Bisikiewicz. 1983. xe2x80x9cResults of bacteriophage treatment of suppurative bacterial infections. I. General evaluation of the results.xe2x80x9d Archivum, Immunol. Therapiae Experimental, 31:267-291; Slopek, S., B. Weber-Dabrowska, M. Dabrowski, and A. Kucharewicz-Krukowska. 1987. xe2x80x9cResults of bacteriophage treatment of suppurative bacterial infections in the years 1981-1986xe2x80x9d, Archivum Immunol. Therapiae Experimental, 35:569-83.
Infections treated with bacteriophage included osteomyelitis, sepsis, empyema, gastroenteritis, suppurative wound infection, pneumonia and dermatitis. Pathogens involved included Staphylococci, Sreptococci, Klebsiella, Shigella, Salmonella, Pseudomonas, Proteus and Escherichia. These articles reported a range of success rates for phage therapy between 80-95% with only rare reversible allergic or gastrointestinal side effects. These results indicate that bacteriophage may be a useful adjunct in the fight against bacterial diseases. However, this literature does not describe, in any way anticipate, or otherwise suggest the use of bacteriophage to modify the composition of colonizing bacterial flora in humans, thereby reducing the risk of subsequent development of active infections.
Salmonella in Humans
Salmonella are the leading cause of food-borne disease in the United States. In 1993, USDA estimated that there were between 700,000 and 3.8 million Salmonella cases in this country, with associated medical costs and productivity losses of between $600 million and $3.5 billion. See Food Safety and Inspection Service, 1995; 9 CFR Part 308; Pathogen Reduction; Hazard Analysis and Critical Control Point (HACCP) Systems; Proposed Rule 60 Fed. Reg. 6774-6889; FoodNet, unpublished data. More exact estimates of incidence have come from CDC""s FoodNet system, based on active surveillance data from seven sentinel sites, with the most recent data suggesting that there are 1.4 million cases annually. See Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S. Bresee, C. Shapiro, P. M. Griffin, and R. V. Tauxe xe2x80x9cFood-related illness and death in the United Statesxe2x80x9d Emerg. Infec. Dis. 5:607-625 (1999). While all Salmonella appear to be able to cause illness, S. typhimurium and S. enteritidis accounted for 22.6% and 22% of all human cases, respectively, in the United States between 1991 and 1995. See Centers for Disease Control and Prevention xe2x80x9cSalmonella Surveillance, Annual Summaryxe2x80x9d 1991, 1992, 1993-1995.
S. typhimurium has become of particular concern because of the recent emergence of a highly antibiotic resistant strain (resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline) designated as definitive type 104 (DT104). In 1979-80, this resistance pattern was seen in 0.6% of S. typhimurium isolates; by 1996, 34% of all U.S. isolates tested by public health laboratories had this pattern, with further testing showing that approximately 90% of these resistant isolates were DT104. See Glynn, M. K., C. Bopp, W. DeWitt, P. Dabney, M. Mokhtar, and F. J. Angulo xe2x80x9cEmergence of multidrug-resistant Salmonella enterica serotype typhimurium DT104 infections in the United Statesxe2x80x9d N. Eng. J. Med. 19:1333-8 (1988). Recent data also suggest that DT-104 is beginning to acquire resistance to trimethoprim and quinolones. See Wall, P. G., D. Morgan, K. Lamden. M. Ryan, M. Griffin, E. J. Threlfall, L. R. Ward, and B. Rowe xe2x80x9cA case control study of infection with an epidemic strain of multiresistant Salmonella typhimurium DT104 in England and Walesxe2x80x9d Commun. Dis. Rep. CDR Rev. 4:R130-8135 (1994). While data on pathogenicity are limited, DT104 appears to be responsible for increased human morbidity and mortality, as compared with other Salmonella. See Centers for Disease Control xe2x80x9cMultidrug resistant Salmonella serotype typhimuriumxe2x80x94United States, 1996xe2x80x9d Morbid Mortal Weekly Rep. 46:308-10 (1997).
Among S. enteritidis isolates, attention has focused on phage types 8 and 4. Phage type 8 accounts for approximately half of all U.S. S. enteritidis isolates. See Hickman-Brenner, F. W., A. D. Stubbs, and J. J. Farmer, III xe2x80x9cPhage typing of Salmonella enteritidis in the United Statesxe2x80x9d J. Clin. Microbiol., 29;2817-23 (1991); Morris, J. G., Jr., D. M. Dwyer, C. W. Hoge, A. D. Stubbs, D. Tilghman, C. Groves, E. Israel, and J. P. Libonati xe2x80x9cChanging clonal patterns of Salmonella enteritidis in Maryland: An evaluation of strains isolated between 1985-90xe2x80x9d J. Clin. Microbiol., 30:1301-1303 (1992). Phage type 4 is seen less frequently, but has been associated with recent major outbreaks; it clearly has increased virulence in chickens, and, again, may have increased virulence in humans. See Humphrey T. J., Williams A., McAlpine K., Lever M. S., Guard-Petter J., and J. M. Cox xe2x80x9cIsolates of Salmonella enterica Enteritidis PT4 with enhanced heat and acid tolerance are more virulent in mice and more invasive in chickensxe2x80x9d Epidemiol. Infect. 117:79-88 (1996); Rampling, A., J. R. Anderson, R. Upson, E. Peters, L. R. Ward, and B. Rowe xe2x80x9cSalmonella enteritidis phage type 4 infection of broiler chickens: a hazard to public healthxe2x80x9d Lancet, ii:436-8 (1989).
In healthy adults, Salmonella generally causes a self-limited diarrheal illness; however, these individuals may asymptomatically carry the organism in their intestinal tract for six months or more after cessation of symptoms (convalescent carriage), serving as one source for continue transmission of the organism in the community. The elderly, the very young, and persons who are immunocompromised are at risk for Salmonella bacteremia, which may occur in as many as 5% of infected xe2x80x9chigh riskxe2x80x9d patients. See Taylor, J. L., D. M. Dwyer, C. Groves, A. Bailowitz, D. Tilghman, V. Kim, A. Joseph, and J. G. Morris, Jr. xe2x80x9cSimultaneous outbreak of Salmonella enteritidis and Salmonella schwarzengrund in a nursing home: association of S. enteritidis with bacteremia and hospitalizationxe2x80x9d J. Infect. Dis. 167:781-2 (1993). Between 1% and 3% of infected persons may also develop reactive arthritis, with the possibility of associated long-term disability.
Antibiotic therapy of diarrheal illness is not effective, and may actually prolong intestinal carriage. See Alavidze, Z., and I. Okolov xe2x80x9cUse of specific bacteriophages in prophylaxis of intrahospital infections caused by P. aeruginosaxe2x80x9d In: Abst., All-Soviet Union conference xe2x80x9cModern biology at the service of public health,xe2x80x9d Kiev, Ukraine (1988). Bacteremia is, obviously, treated with antibiotics, although the emergence of highly resistant strains such as DT104 has begun to create problems in patient management. See Wail, P. G., D. Morgan, K. Lamden, M. Ryan, M. Griffin, E. J. Threlfall, L. R. Ward, and B. Rowe xe2x80x9cA case control study of infection with an epidemic strain of multiresistant Salmonella typhimurium DT104 in England and Walesxe2x80x9d Commun. Dis. Rep. CDR Rev. 4-R130-RI35 (1994). There is currently no effective means of limiting or eradicating carriage of the organism in the intestinal tract. See Neill, M. A., S. M. Opal, J. Heelan, R. Giusti, J. E. Cassidy, R. White, and K. H. Mayer xe2x80x9cFailure of ciprofloxacin to eradicate convalescent fecal excretion after acute Salmonellosis: experience during an outbreak in health care workersxe2x80x9d Ann. Intern Med. 119:195-9 (1991).
Salmonella in Chickens
USDA estimates that in 50-75% of human Salmonella cases the microorganism is acquired from meat, poultry, or eggs, with poultry serving as the primary vehicle of transmission. Salmonella are part of the normal, colonizing intestinal flora in many animals, including chickens. Studies conducted in the early 1990""s by USDA indicated that 20-25% of broiler carcasses and 18% of turkey carcasses were contaminated with Salmonella prior to sale. See Food Safety and Inspection Service (1995); 9 CFR Part 308; Pathogen Reduction; Hazard Analysis and Critical Control Point (HACCP) Systems; Proposed Rule; 60 Fed. Reg. 6774-6889.
Contamination may result from rupture of the intestinal tract during slaughter. However, with current slaughter techniques, removal of the viscera seldom results in intestinal rupture and carcass contaminationxe2x80x94and, when it does occur, the carcass is immediately tagged for xe2x80x9creprocessing.xe2x80x9d The more common source of Salmonella is the skin of the animal itself, with the feather follicles serving as a sanctuary for bacteria. In contrast to beef, chickens are slaughtered xe2x80x9cskin on,xe2x80x9d so that antemortem contamination of feathers becomes an important element in determining whether Salmonella can be isolated from the carcass. The close quarters in chicken houses, and the piling of chicken crates on trucks on the way to slaughterhouses, results in frequent contamination of feathers by feces. If members of a flock have high levels of intestinal colonization with Salmonella, there are multiple opportunities for contamination of feathers and feather follicles with the microorganism, and, in turn, for Salmonella contamination of the final product.
According to the CDC FoodNet/Salmonella surveillance system, the five most common human Salmonella isolates in the United States during 1990-1995 were S. typhimurium, S. enteritidis, S. heidelberg, S. newport, and S. hadar. Further, according to the USDA/FSIS data, the five most common Salmonella serotypes isolated from broiler chickens during the same period were S. heidelberg, S. kentucki, S. hadar, S. typhimurium, and S. thomson. While Applicants do not consider this to be an exhaustive list, Applicants note that these are common Salmonella isolates and serotypes.
The rate of Salmonella contamination of poultry carcasses was a major focus of the recently implemented revision of the national food safety regulations (Pathogen Reduction; Hazard Analysis and Critical Control Point (HACCP) Systems), which mandates government testing for Salmonella in all slaughter plants. Regulations now in effect require that product be tested by putting a whole chicken carcass in a xe2x80x9cbaggiexe2x80x9d with culture media and shaking; growth of any Salmonella from broth counts as a positive test. Plants must meet specific standards for percentage of product contaminated, based on national averages; failure to meet these standards results in plant closure. See Food Safety Inspection Service (1996); 9 CFR Part 304, et seq.; Pathogen Reduction; Hazard Analysis and Critical Control Point (HACCP) Systems; Final Rule 61 Fed. Reg. 38806-989. Concerns about Salmonella contamination have also become a major issue in international trade, with Russia and other countries having embargoed millions of dollars worth lots of chickens because of identification of Salmonella in the product.
In this environment, there are strong public health, regulatory, and trade incentives for producers to reduce levels of Salmonella contamination in poultry. Irradiation of raw product (i.e., chicken carcasses) is efficacious, but expensive, and is limited by the small number of irradiation facilities and by consumer acceptance. Treatment of chickens with antibiotics does not eradicate colonization, tending simply to select out for more resistant organisms. Antibiotics (in contrast to phage) generally have activity against multiple bacterial species; their administration can result in serious perturbations in the microbial ecology of the animal""s intestinal tract, with accompanying loss of xe2x80x9ccolonization resistancexe2x80x9d and overgrowth of microorganisms that are resistant to the antimicrobial agent used. Vaccination is similarly ineffective in elimination of Salmonella. See Hassan, J. O., and R. Curtiss, III xe2x80x9cEfficacy of a live avriulent Salmonella typhimurium vaccine in preventing colonization and invasion of laying hens by Salmonella typhimurium and Salmonella enteritidisxe2x80x9d Avian. Dis. 41:783-91 (1997); Methner, U., P. A. Barrow, G. Martin, and H. Meyer xe2x80x9cComparative study of the protective effect against Salmonella colonization in newly hatched SPF chickens using live, attenuated Salmonella vaccine strains, wild-type Salmonella strains or a competitive exclusion productxe2x80x9d Int. J Food Microbiol., 35:223-230 (1997); Tan, S., C. L. Gyles, and B. N. Wilkie xe2x80x9cEvaluation of an aroA mutant Salmonella typhimurium vaccine in chickens using modified semisolid Rappaport Vassiliadis medium to monitor fecal sheddingxe2x80x9d Vet. Microbiol., 54:247-54 (1997).
Competitive exclusion (i.e., administration ofxe2x80x9cgoodxe2x80x9d bacteria to xe2x80x9ccrowd outxe2x80x9d Salmonella and other xe2x80x9cbadxe2x80x9d bacteria) has shown variable success. See Palmu, L, I. Camelin xe2x80x9cThe use of competative exclusion in broilers to reduce the level of Salmonella contamination on the farm and at the processing plantxe2x80x9d Poultry Sci. 76:1501-5 (1997). There is now a commercially available competitive exclusion product, PreEmpt (produced by MS Bioscience), that consists of 27 different bacteria strains- In preliminary testing, it appears to be effective in limiting Salmonella colonization, but its usage is hampered by the cost. Most importantly, its efficacy is significantly decreased if antibiotics are administered to animals as growth additives (a standard practice in the poultry industry).
In the absence of any other definitive means of eradicating the organism, USDA has articulated the concept of Salmonella control through a xe2x80x9cmultiple hurdlexe2x80x9d approach, encouraging implementation of procedures to reduce the risk of contamination during slaughter while at the same time seeking to limit colonization/contamination of broiler flocks by the organism. Under these circumstances, there is a clear market for products and approaches that can be used as part of an overall program of Salmonella control. Any such product should be cheap, safe, and easy to use-, there would also be potential advantages for products which could be targeted toward specific pathogens, such as S. enteritidis PT4 and S. typhimurium DT104.
According to one embodiment of the present invention, a method for sanitation using at least one bacteriophage is disclosed. The method includes the steps of (1) storing the at least one bacteriophage in a container; and (2) applying the at least one bacteriophage to a surface to be sanitized with a dispersing mechanism.
The container may be, inter alia, a pressurized container (e.g., a aerosol canister), may be a fogging device; may be a trigger spray device; or may be a pump spray device. The bacteriophage may be poured, brushed, wiped, painted, or coated on the area or an object. The bacteriophage may be transferred from a transfer vehicle, which may be a towel, a sponge, a roller, a paper product, a towelette, etc., to the area or object. In one embodiment, hoses or sprinklers may be used. Once applied, the area or object may be flushed with water.
The areas or objects that may have the bacteriophage applied include, inter alia, livestock pens, live stock feeding areas, live stock slaughter areas, live stock waste areas, knives, shovels, rakes, saws, livestock handling devices, hospital rooms, operating rooms, bathrooms, waiting rooms, beds, chairs, wheel chairs, gurneys, surgical tables, operating room floors, operating room walls, surfaces in an intensive care unit, electrocardiographs, respirators, cardiovascular assist devices, intraaortic balloon pumps, infusion devices, other patient care devices, televisions, monitors, remote controls, and telephones. The present invention may be used to decontaminate military equipment, including aircraft, vehicles, electronic equipment, and weapons.
According to another embodiment of the present invention, a sanitation device that dispenses at least one bacteriophage is disclosed. The device includes a container, at least one bacteriophage stored in the container, and a dispersing mechanism that disperses the at least one bacteriophage from the container.
According to another embodiment of the present invention, a method for poultry processing sanitation with at least one bacteriophage is disclosed. The method includes the step of applying at least one bacteriophage to fertilized eggs.
According to another embodiment of the present invention, a method for poultry processing sanitation with at least one bacteriophage is disclosed. The method includes the step of applying at least bacteriophage to at least one freshly-hatched bird.
According to another embodiment of the present invention, a method for poultry processing sanitation with at least one bacteriophage is disclosed. The method includes the step of providing drinking water containing at least bacteriophage.
According to another embodiment of the present invention, a method for poultry processing sanitation with at least one bacteriophage is disclosed. The method includes the step of providing food with the at least bacteriophage.
According to another embodiment of the present invention, a method for poultry processing sanitation with at least one bacteriophage is disclosed. The method includes the step of applying at least one bacteriophage to post-chill birds.
Developing novel methodologies/antimicrobials for reducing poultry contamination with Salmonella may be expected to have tremendous impact on human health; these antimicrobials also may have utility in managing infections caused by multi drug-resistant Salmonella (e.g., DT104) strains. It is an object of this invention to isolate and characterize phages that may have utility in managing Salmonella infections. The present inventors have isolated several bacteriophages active against genetically diverse Salmonella strains, and have demonstrated the utility of these phages in cleaning Salmonella contaminated surfaces. These phages may be used in managing Salmonella-contamination and prophylaxis/treatment of diseases caused by Salmonella, including multidrug resistant DT-104 strains.
One attractive modality to control the rates of Salmonella contamination of poultry is to use Salmonella-specific bacteriophages. Bacteriophages are specific for prokaryotes, and they are highly selective for a bacterial species or serotype (i.e., they permit targeting of specific bacteria, without disrupting normal flora). In addition, phages are relatively, easy to propagate and purify on a production scale. Furthermore, extensive studies in the Soviet Union and several Eastern European countries have demonstrated the safety and efficacy of bacteriophage therapy for many bacterial diseases. Extending the concept of phage treatment to the primary prevention of salmonellosis, by (i) administering specific phages to chickens, and (ii) using phages for environmental clean-up of chicken houses, processing plants, etc., may reduce or eliminate Salmonella strains which ate of ma or public health significance.
According to another embodiment of the present invention, a method for foodstuff packaging is disclosed. The method includes the steps of (1) providing foodstuff for packaging: (2) applying at least one bacteriophage to the foodstuff; and (3) packaging the foodstuff with a packaging material.
According to another embodiment of the present invention, a method for foodstuff packaging is disclosed. The method includes the steps of (1) providing a package containing the foodstuff; and (2) inserting a matrix containing at least one bacteriophage into the package.
According to another embodiment of the present invention, a method for foodstuff packaging is disclosed. The method includes the steps of (1) providing a foodstuff; (2) providing a packaging material comprising at least one bacteriophage; and (3) packaging the foodstuff with the packaging material.
According to another embodiment of the present invention, a method for foodstuff sanitation with at least one bacteriophage is disclosed. The method includes the steps of (1) providing a foodstuff; and (2) applying the at least one bacteriophage to the foodstuff.
According to another embodiment of the present invention, a method for decontamination using at least one bacteriophage is disclosed. The method includes the step of applying at least one bacteriophage to an area contaminated with at least one pathogenic bacteria.